Preprint volume - SIBM

Pre-print Volume - Posters
Topic 1: BIODIVERSITY AND CONSERVATION SCIENCE: CONTRIBUTING TO MANAGEMENT
41 st S.I.B.M. CONGRESS Rapallo (GE), 7-11 June 2010
87
Fig. 1 – Sampling locations of Calderón
et al. (2006) (paddle) and of the present
work (arrow).
Località di campionamento di Calderón
et al. (2006) (pagaia) e del presente
lavoro (freccia).
Each sample consisted of a 6-8 cm long fragment collected from the tip of a colony and
preserved in 96% ethanol. Sampling was carried out only on those gorgonians that
were at least 50 cm high. DNA was extracted from 20 polyps of each fragment using a
Salting Out protocol modified from Aljanabi & Martinez (1997). The new protocol
employed a highly concentrated lysing solution (EDTA 100 mM; Tris HCl 10 mM pH
7.5; SDS 0.6%; NaCl 400 mM) in which tissue was incubated overnight. DNA was
PCR-amplified using the COI primers reported in Calderón et al. (2006).
(COI Cni F: 5’-GGYACTYTATATTTACTATTTGG-3’; COI Cni R: 5’-
CCSGCAGGATCAAAGAAWGTTG-3’). Amplified products were then purified and
sequenced by a commercial company. Two individuals were sequenced from each
location and the obtained nucleotide sequences aligned using the software BioEdit
vers. 7.0.9.0 (Hall, 1999).
Results – PCR amplifications delivered fragments of 545 bp size. Nucleotide
sequences were inserted in GenBank and they confirmed amplification of the COI
region. All obtained sequences were identical to each other and to the one of P. clavata
deposited in GenBank by Calderón et al. (2006) (GenBank access n°: AY827539.1).
Conclusions – The genetic homogeneity at the COI locus of P. clavata, in individuals
sampled at locations that were several hundred kilometers distant, confirms Calderón et
al.’s (2006) results even after enlargement of the study area and use of a higher number
of individual colonies. The lack of polymorphism at the COI region in P. clavata could
be attributed to a slow rate of mutation of the gene or efficient repairing system of this
species’ mtDNA. To fully accomplish the objective of this work the characterization of
genetic markers with a sufficient degree of polymorphism is needed.
References
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genomic DNA for PCR-based techniques. Nucleic Acids Research, 25: 4692-4693.
CALDERÓN I., GARRABOU J., AURELLE D. (2006) – Evaluation of the utility of COI and ITS
markers as tools for population genetic studies of temperate gorgonians. Journal of Experimental
Marine Biology and Ecology, 336: 184-197.
CARPINE C., GRASSHOF M. (1975) – Les Gorgonaires de la Méditerranée. Bulletin de l'Institut
Océanographique, 71: 1-140.
CUPIDO R., COCITO S., BORDONE A., SGORBINI S., SANTANGELO G. (2008) – Response of
a gorgonian (Paramuricea clavata) population to mortality events: recovery or loss? Aquatic
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HALL T.A. (1999) – BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.